Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage
Tóm tắt
A major hurdle to transcriptome profiling by deep-sequencing technologies is that abundant transcripts, such as rRNAs, can overwhelm the libraries, severely reducing transcriptome-wide coverage. Methods for depletion of such unwanted sequences typically require treatment of RNA samples prior to library preparation, are costly and not suited to unusual species and applications. Here we describe Probe-Directed Degradation (PDD), an approach that employs hybridisation to DNA oligonucleotides at the single-stranded cDNA library stage and digestion with Duplex-Specific Nuclease (DSN). Targeting Saccharomyces cerevisiae rRNA sequences in Illumina HiSeq libraries generated by the split adapter method we show that PDD results in efficient removal of rRNA. The probes generate extended zones of depletion as a function of library insert size and the requirements for DSN cleavage. Using intact total RNA as starting material, probes can be spaced at the minimum anticipated library size minus 20 nucleotides to achieve continuous depletion. No off-target bias is detectable when comparing PDD-treated with untreated libraries. We further provide a bioinformatics tool to design suitable PDD probe sets. We find that PDD is a rapid procedure that results in effective and specific depletion of unwanted sequences from deep-sequencing libraries. Because PDD acts at the cDNA stage, handling of fragile RNA samples can be minimised and it should further be feasible to remediate existing libraries. Importantly, PDD preserves the original RNA fragment boundaries as is required for nucleotide-resolution footprinting or base-cleavage studies. Finally, as PDD utilises unmodified DNA oligonucleotides it can provide a low-cost option for large-scale projects, or be flexibly customised to suit different depletion targets, sample types and organisms.
Tài liệu tham khảo
Mercer TR, Gerhardt DJ, Dinger ME, Crawford J, Trapnell C, Jeddeloh JA, Mattick JS, Rinn JL: Targeted RNA sequencing reveals the deep complexity of the human transcriptome. Nat Biotechnol. 2012, 30: 99-104.
Ingolia NT, Ghaemmaghami S, Newman JRS, Weissman JS: Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science. 2009, 324: 218-223. 10.1126/science.1168978.
Ingolia NT, Brar GA, Rouskin S, McGeachy AM, Weissman JS: The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat Protoc. 2012, 7: 1534-1550. 10.1038/nprot.2012.086.
Ule J, Jensen K, Mele A, Darnell RB: CLIP: a method for identifying protein–RNA interaction sites in living cells. Methods. 2005, 37: 376-386. 10.1016/j.ymeth.2005.07.018.
Licatalosi DD, Mele A, Fak JJ, Ule J, Kayikci M, Chi SW, Clark TA, Schweitzer AC, Blume JE, Wang X, Darnell JC, Darnell RB: HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature. 2008, 456: 464-469. 10.1038/nature07488.
Murigneux V, Saulière J, Roest Crollius H, Le Hir H: Transcriptome-wide identification of RNA binding sites by CLIP-seq. Methods. 2013, 63: 32-40. 10.1016/j.ymeth.2013.03.022.
Adiconis X, Borges-Rivera D, Satija R, DeLuca DS, Busby MA, Berlin AM, Sivachenko A, Thompson DA, Wysoker A, Fennell T, Gnirke A, Pochet N, Regev A, Levin JZ: Comparative analysis of RNA sequencing methods for degraded or low-input samples. Nat Methods. 2013, 10: 623-629. 10.1038/nmeth.2483.
Morlan JD, Qu K, Sinicropi DV: Selective depletion of rRNA enables whole transcriptome profiling of archival fixed tissue. PLoS ONE. 2012, 7: e42882-10.1371/journal.pone.0042882.
Casey J, Davidson N: Rates of formation and thermal stabilities of RNA: DNA and DNA: DNA duplexes at high concentrations of formamide. Nucleic Acids Res. 1977, 4: 1539-1552. 10.1093/nar/4.5.1539.
Ko MSH: An “equalized cDNA library” by the reassociation of short double-stranded cDNAs. Nucleic Acids Res. 1990, 18: 5705-5711. 10.1093/nar/18.19.5705.
Yi H, Cho Y-J, Won S, Lee J-E, Jin Yu H, Kim S, Schroth GP, Luo S, Chun J: Duplex-specific nuclease efficiently removes rRNA for prokaryotic RNA-seq. Nucleic Acids Res. 2011, 39: e140-10.1093/nar/gkr617.
VanderNoot VA, Langevin SA, Solberg OD, Lane PD, Curtis DJ, Bent ZW, Williams KP, Patel KD, Schoeniger JS, Branda SS, Lane TW: cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications. BioTech. 2012, 53: 373-380.
Bogdanova EA, Shagina IA, Mudrik E, Ivanov I, Amon P, Vagner LL, Lukyanov SA, Shagin DA: DSN depletion is a simple method to remove selected transcripts from cDNA populations. Mol Biotechnol. 2009, 41: 247-253. 10.1007/s12033-008-9131-y.
Allawi HT, SantaLucia J: Thermodynamics and NMR of Internal G · T Mismatches in DNA. Biochemistry. 1997, 36: 10581-10594. 10.1021/bi962590c.
Engel SR, Dietrich FS, Fisk DG, Binkley G, Balakrishnan R, Costanzo MC, Dwight SS, Hitz BC, Karra K, Nash RS, Weng S, Wong ED, Lloyd P, Skrzypek MS, Miyasato SR, Simison M, Cherry JM: The reference genome sequence of saccharomyces cerevisiae: then and now. G3 (Bethesda). 2014, 4: 389-398. 2014.
Langmead B, Salzberg SL: Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012, 9: 357-359. 10.1038/nmeth.1923.
Lamm AT, Stadler MR, Zhang H, Gent JI, Fire AZ: Multimodal RNA-seq using single-strand, double-strand, and CircLigase-based capture yields a refined and extended description of the C. elegans transcriptome. Genome Res. 2011, 21: 265-275. 10.1101/gr.108845.110.
Levin JZ, Yassour M, Adiconis X, Nusbaum C, Thompson DA, Friedman N, Gnirke A, Regev A: Comprehensive comparative analysis of strand-specific RNA sequencing methods. Nat Methods. 2010, 7: 709-715. 10.1038/nmeth.1491.
Shagin DA: A novel method for SNP detection using a new duplex-specific nuclease from crab hepatopancreas. Genome Res. 2002, 12: 1935-1942. 10.1101/gr.547002.
Giannoukos G, Ciulla DM, Huang K, Haas BJ, Izard J, Levin JZ, Livny J, Earl AM, Gevers D, Ward DV, Nusbaum C, Birren BW, Gnirke A: Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes. Genome Biol. 2012, 13: r23-10.1186/gb-2012-13-3-r23.
Behm Ansmant I, Helm M, Motorin Y: Use of specific chemical reagents for detection of modified nucleotides in RNA. J Nucleic Acids. 2011, 2011: 1-17.
Squires JE, Patel HR, Nousch M, Sibbritt T, Humphreys DT, Parker BJ, Suter CM, Preiss T: Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA. Nucleic Acids Res. 2012, 40: 5023-5033. 10.1093/nar/gks144.
Anisimova VE, Rebrikov DV, Shagin DA, Kozhemyako VB, Menzorova NI, Staroverov DB, Ziganshin R, Vagner LL, Rasskazov VA, Lukyanov SA, Shcheglov AS: Isolation, characterization and molecular cloning of Duplex-Specific Nuclease from the hepatopancreas of the Kamchatka crab. BMC Biochem. 2008, 9: 14-10.1186/1471-2091-9-14.
Nilsen IW, Øverbø K, Jensen Havdalen L, Elde M, Gjellesvik DR, Lanes O: The enzyme and the cDNA sequence of a thermolabile and double-strand specific DNase from Northern Shrimps (Pandalus borealis). PLoS ONE. 2010, 5: e10295-10.1371/journal.pone.0010295.
Mastrokolias A, den JT D, van Ommen GB, Hoen PAC ‘t, van Roon-Mom WMC: Increased sensitivity of next generation sequencing-based expression profiling after globin reduction in human blood RNA. BMC Genomics. 2012, 13: 28-10.1186/1471-2164-13-28.